1. Field of the Invention
The invention in the fields of molecular biology and medicine relates to a method for detecting or screening for mutations or heterozygosity involving as little as one base change or a single base addition to, or deletion from, the wild-type DNA sequence, as well as methods for removing mismatch-containing DNA from batches of amplified DNA.
2. Description of the Background Art
Progress in human molecular and medical genetics depends on the efficient and accurate detection of mutations and sequence polymorphisms, the vast majority of which results from single base substitutions and small additions or deletions. Assays capable of detecting the presence of a particular mutation or mutant nucleic acid sequence in a sample are therefore of substantial importance in the prediction and diagnosis of disease, forensic medicine, epidemiology and public health. Such assays can be used, for example, to detect the presence of a mutant gene in an individual, allowing determination of the probability that the individual will suffer from a genetic disease. The ability to detect a mutation has taken on increasing importance in early detection of cancer or discovery of susceptibility to cancer with the discovery that discrete mutations in cellular oncogenes can result in activation of that oncogene leading to the transformation of that cell into a cancer cell.
The desire to increase the utility and applicability of such assays is often frustrated by assay sensitivity as well as complexity and cost. Hence, it would be highly desirable to develop more sensitive as well as simple and relatively inexpensive assays for detection of alterations in DNA.
Nucleic acid detection assays can be based on any of a number of characteristics of a nucleic acid molecule, such as its size, sequence, susceptibility to digestion by restriction endonucleases, etc. The sensitivity of such assays may be increased by altering the manner in which detection is reported or signaled to the observer. Thus, for example, assay sensitivity can be increased through the use of detectably labeled reagents such as enzymes (Kourilsky et al., U.S. Pat. No. 4,581,333), radioisotopes (Falkow et al., U.S. Pat. No. 4,358,535; Berninger, U.S. Pat. No. 4,446,237), fluorescent labels (Albarella et al., EP 144914), chemical labels (Sheldon III et al., U.S. Pat. No. 4,582,789; Albarella et al., U.S. Pat. No. 4,563,417), modified bases (Miyoshi et al., EP 119448), and the like.
Most methods devised to attempt to detect genetic alterations consisting of one or a few bases involve hybridization between a standard nucleic acid (DNA or RNA) and a test DNA such that the mutation is revealed as a mispaired or unpaired base in a heteroduplex molecule. Detection of these mispaired or unpaired bases has been accomplished by a variety of methods (Myers, R. M. et al., Cold Spring Harbor Symp. Quant. Biol. 51:275-284 (1986); Gibbs, R. et al., Science 236:303-305 (1987); Lu, A. S. et al., 1992, Genomics 14:249-255 (1992); Cotton, R. G. et al., Proc. Natl. Acad. Sci. USA 85:4397-4401 (1988); Cotton, R. G., Nucl. Acids Res 17:4223-4233 (1989)). Major problems of various of these methods include a requirement for using RNA or an ability to detect only a limited number of mismatches (though the Cotton et al. method is said to detect all possible single base pair mismatches). These methods are also generally laborious, require that the exact location of the mutation be known and are difficult to interpret when the sample DNA is heterozygous for the mutation in question. Therefore, they are not practical for use in screening for polymorphisms. Publications from Caskey's laboratory (Caskey, C. T. et al., European Patent Publication 333,465 (Sep. 20, 1989); Grompe, M. et al., Proc. Natl. Acad. Sci. USA 86:5888-5892 (1989)) disclose a method for localizing a mutation using PCR-amplified cDNA.
Wagner et al. (PCT publication WO93/02216, disclosing an invention made by the present inventor) shows that E. coli MutS, in solution, detects G//T, G//G and A//C mismatches as well as a frameshift mutation of +1 base. There is no disclosure of using immobilized MutS in this method. (The symbol "//" is used herein with nucleotide base designations to indicate mispaired bases. Proper Watson-Crick base pairs are designated with a ":" for example G:C.)
Lishanskaya et al. (Human Genet. 51,(4 Suppl):A385, abstract #1517 (1992)) is an abstract disclosing the detection of heterozygotes for the amplified CFTR (cystic fibrosis) gene. The method was not shown to be useful for detecting single base mismatches, or mispairings of 1, 2 or 4 bases. Rather, this reference disclosed detection of only one specific mutation of one type caused by a three base pair deletion. The detection was achieved by a gel mobility band-shift assay of MutS-heteroduplex complexes. This reference did not suggest immobilizing a MBP. Lishanski, A. et al., Proc. Natl. Acad. Sci. USA 91:2674-2678 (1994 March) described detection of frame shift mutations and A//C, G//T, G//A and T//C mismatches (though primarily G//T) using MutS. Significantly, the method worked poorly with duplexes containing single base pair mismatches. At the time this paper was published (after the filing date of the priority application of the present application), Lishanski et al. were still trying to develop improved detection methods (page 2677 column 2, end of "Conclusions" section, referring to "preliminary experiments") such as the very methods that the present inventor had already invented and disclosed in U.S. Ser. No. 08/147,785). Lishanski et al. (supra) thereby documents (1) the recognition in the art of the need or desire for the present invention and (2) the fact that the present methods were not yet available to the public (as of the date this paper was submitted, Dec. 17, 1993).
A number of publications describe methods for studying certain DNA binding proteins, in particular sequence-specific transcription factors, by electrophoretic separation of generally under denaturing conditions, blotting ("Southwestern blots") or spotting of the separated proteins onto nitrocellulose filters, and probing of the filters with labeled DNA or oligonucleotides. See, for example, Bowen, B. et al., Nucl. Acids Res. 8:1-20 (1979); Miskimins, W. K. et al., Proc. Natl. Acad. Sci. USA 82:6741-6744 (1985); Keller, A. D. et al., Nucl. Acids Res. 19:4675-80 (1991); Norby, P. L. et al., Nucl. Acids Res. 20:6317-6321 (1992)). Such initially denatured proteins must be renatured to perform their DNA binding functions; many of these proteins cannot be successfully renatured.
Similar approaches have been used to screen expression libraries by preparing protein replica filters and probing them with labeled DNA or oligonucleotides (Singh et al., Cell 52:415-423 (1988); Singh et al., BioTechniques 7:253-261 (1989); Vinson et al., Genes & Devel. 2:801-806 (1988)). Oliphant, A. R. et al. (Molec. Cell. Biol. 9:2944-2949 (1989)) reported use of a sequence-specific DNA binding protein (yeast GCN4 transcriptional activator) coupled to Sepharose to select DNA molecules containing binding sites for this protein from random sequence oligonucleotides. Interactions of DNA with immobilized E. coli single stranded DNA-binding protein (SSB) has been reported (Perrino, F. W. et al., J. Biol. Chem. 263:11833-11839 (1988)) and is reviewed in Meyer, R. R. et al. (Microbiol. Rev. 54:342-380 (1990)).